Estrogens, a family of related molecules that stimulate the development and maintenance of female characteristics and sexual reproduction, act on target tissues by binding to estrogen receptors. Some drugs that block the action of estrogen in certain tissues actually can mimic the action of estrogen in other tissues. Such selectivity is made possible by the fact that the estrogen receptors (ERs) of different target tissues vary in chemical structure. There are two ERs: the ERα is found in the endometrium, breast cancer cells and ovarian stroma cells, while the ERβ is found in kidney, brain, bone, heart, lungs, intestinal mucosa, prostate, and endothelial cells. Some estrogenic or estrogen-like compounds may have different binding affinities for ERα and ERβ. These differences allow estrogen-like drugs to interact in different ways with the estrogen receptors of different tissues, namely, they can selectively activate (or block) one type of ER or to promote ER interactions with different proteins such as transcriptional co-activator or co-repressor proteins. Such drugs are called selective estrogen receptor modulators, or SERMs, because they selectively stimulate or inhibit the estrogen receptors of different target tissues.
Isoflavones are a class of phytoestrogen plant-derived compounds, which exhibit multiple biological effects and, in some systems, operate through estrogen receptors, upon which they may act as agonists, antagonists or mixed agonist-antagonists. Soybeans and soy products and red clover are the richest sources of isoflavones in the human diet.
Isoflavones and certain derivatives thereof are known to compete with estrogen on binding to estrogen receptors (ERs), and can thus be used for modulation of ERs functioning. Having the capability to modulate ER activity, isoflavone and derivatives thereof may be useful to treat or prevent a variety of diseases and conditions related to estrogen receptor functioning in mammals, preferably humans. Epidemiological and in vitro and in vivo animal studies indicate that isoflavones are promising agents for cancer chemoprevention and as inhibitors of tumor progression.
The isoflavones that have been most studied are genistein, biochanin A and daidzein. These compounds may act as weak estrogens or as anti-estrogens, depending on the cell type and concentration of isoflavone used. In various estrogen sensitive human cancer cell lines (e.g breast, colon, etc) these isoflavones can act as weak estrogens and stimulate cell growth at concentrations ranging from 0.1 to 20 μM, while inhibiting cell proliferation at concentrations greater than 20 μM. The affinity of most isoflavones to the two subtypes of ER is low (Kuiper et al. 1998), with the exception of genistein, which shows a stronger selectivity for ERβ over ERα. Both genistein and daidzein display 100-fold greater sensitivity (Harris et al., 2005) for activating transcription in transfected cells via ERβ compared to ERα.
The mechanisms underlying the anti-proliferative effects of these compounds vary significantly among the various isoflavones and the type of cell under investigation. For example, inhibition of cell proliferation by isoflavones may involve interference with signaling via the epidermal growth factor receptor kinase (Akiyama et al., 1987), effects on cell cycle (Agarwal 2000), caspases or transforming growth factor β signaling (Kim et al., 1998). Several approaches have been applied in an attempt to utilize and improve the cytotoxic potency of isoflavones. These include targeting of the EGF receptor in breast cancer cells by using a conjugate of genistein coupled to EGF, EGF-Gen (Uckun et al., 1998); generation of synthetic derivatives of chromen-4-one (the minimal structural motif of genistein) complexed to copper (Barve et al., 2006); and reducing the ketone group at position C-4 of daidzein, to yield the dihydro derivative of daidzein phenoxodiol. In in vitro studies, EGF-Gen and the synthetic derivatives of the structurally modified genistein analogue, chromen-4-one complexed to copper, exerted higher cytotoxic activity than genistein, with lower IC50 in breast cancer (Uckun et al., 1998) and pancreatic cancer cells (Sarkar et al., 2006). Phenoxodiol showed strong apoptotic (Kamsteeg et al., 2003) and anti-angiogenic activities (Gamble et al., 2006) in ovarian carcinoma in vitro and reduced tumor volume of ovarian xenografts in vivo (Mor et al., 2006).
Previous studies of the present inventors have shown that a derivative of genistein, 6-carboxymethylene genistein, behaved like a selective estrogen receptor modulator, with unique and differential effects on the vasculature, bone, and uterus (Somjen et al., 2002).
WO 03/079965, of the same applicants, discloses carboxy derivatives of isoflavones such as biochanin A, formononetin, daidzein and genistein, and conjugates thereof with a bioactive moiety, particularly daunomycin. These isoflavone derivatives are said to exhibit selective estrogen receptor modulation, i.e., to display both weak estrogenic and anti-estrogenic properties in various cancer cell lines.
In order to more effectively inhibit cell growth, it is still desirable to develop derivatives of isoflavones exhibiting more potent inhibitory activity than the parent isoflavones, but with only minimal estrogenic activity.